RESUMEN
Helicobacter pylori infection predisposes carriers to a high risk of developing gastric cancer. The cell-of-origin of antral gastric cancer is the Lgr5+ stem cell. Here, we show that infection of antrum-derived gastric organoid cells with H. pylori increases the expression of the stem cell marker Lgr5 as determined by immunofluorescence microscopy, qRT-PCR, and Western blotting, both when cells are grown and infected as monolayers and when cells are exposed to H. pylori in 3D structures. H. pylori exposure increases stemness properties as determined by spheroid formation assay. Lgr5 expression and the acquisition of stemness depend on a functional type IV secretion system (T4SS) and at least partly on the T4SS effector CagA. The pharmacological inhibition or genetic ablation of NF-κB reverses the increase in Lgr5 and spheroid formation. Constitutively active Wnt/ß-catenin signaling because of Apc inactivation exacerbates H. pylori-induced Lgr5 expression and stemness, both of which persist even after eradication of the infection. The combined data indicate that H. pylori has stemness-inducing properties that depend on its ability to activate NF-κB signaling.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , FN-kappa B , Receptores Acoplados a Proteínas G , Neoplasias Gástricas , Vía de Señalización Wnt , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Humanos , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Vía de Señalización Wnt/genética , FN-kappa B/metabolismo , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Organoides/metabolismo , Organoides/microbiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Células Madre/metabolismo , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/genética , Sistemas de Secreción Tipo IV/metabolismo , Sistemas de Secreción Tipo IV/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/microbiología , Estómago/microbiología , Estómago/patologíaRESUMEN
Helicobacter pylori infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild-type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of H. pylori-induced DNA damage and replication stress and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single-molecule DNA fiber assays, and immunofluorescence microscopy of DNA repair foci that H. pylori induces transcription-dependent DNA damage in actively replicating, Leucine-rich-repeat containing G-Protein-Coupled Receptor 5 (Lgr5)-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by Apc inactivation, but not by Trp53 or Smad4 loss, or Erbb2 overexpression. Our data suggest that H. pylori induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Animales , Humanos , Ratones , Daño del ADN , Genes Supresores de Tumor , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Receptores Acoplados a Proteínas G/genética , Células Madre , Neoplasias Gástricas/patologíaRESUMEN
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Humanos , Especies Reactivas de Oxígeno , Fase S/genética , Proteínas de Unión al ADN/metabolismo , Hidroxiurea/farmacología , ADNRESUMEN
R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co-transcriptionally in the opposite orientation to replication fork progression. A recent study has shown that replication forks stalled by co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by ELL-dependent reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds R-loops in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4-ELL pathway for resolution of R-loop-mediated transcription-replication conflicts, which may be involved in R-loop unwinding.
Asunto(s)
Replicación del ADN , Estructuras R-Loop , Humanos , Replicación del ADN/genética , ADN Helicasas/metabolismo , Endonucleasas/metabolismo , ADN/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismoRESUMEN
R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.
Asunto(s)
Inestabilidad Cromosómica/efectos de los fármacos , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Estructuras R-Loop , Recombinasa Rad51 , Línea Celular Tumoral , Humanos , Complejo de Reconocimiento del Origen/metabolismo , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/metabolismoRESUMEN
Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate ß-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of ß-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Helicobacter pylori/patogenicidad , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , ADN/química , ADN/genética , Daño del ADN , Replicación del ADN/efectos de los fármacos , Floxuridina , Glicosiltransferasas/metabolismo , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Interacciones Huésped-Patógeno/fisiología , Humanos , Lipopolisacáridos/metabolismo , Mutación , FN-kappa B/genética , Proteínas Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patologíaRESUMEN
Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.
Asunto(s)
Replicación del ADN/fisiología , Estructuras R-Loop/genética , Recombinasa Rad51/metabolismo , Línea Celular Tumoral , ADN Ligasas/metabolismo , ADN Polimerasa III/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Células HeLa , Humanos , Estructuras R-Loop/fisiología , Recombinasa Rad51/genética , Recombinasa Rad51/fisiología , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , RecQ Helicasas/metabolismo , RecQ Helicasas/fisiología , Transcripción Genética/genéticaRESUMEN
Sensing of cytoplasmic DNA by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) results in production of the dinucleotide cGAMP and consecutive activation of stimulator of interferon genes (STING) followed by production of type I interferon (IFN). Although cancer cells contain supra-normal concentrations of cytoplasmic DNA, they rarely produce type I IFN spontaneously. This suggests that defects in the DNA-sensing pathway may serve as an immune escape mechanism. We find that cancer cells produce cGAMP that is transferred via gap junctions to tumor-associated dendritic cells (DCs) and macrophages, which respond by producing type I IFN in situ. Cancer-cell-intrinsic expression of cGAS, but not STING, promotes infiltration by effector CD8+ T cells and consequently results in prolonged survival. Furthermore, cGAS-expressing cancers respond better to genotoxic treatments and immunotherapy. Thus, cancer-cell-derived cGAMP is crucial to protective anti-tumor CD8+ T cell immunity. Consequently, cancer-cell-intrinsic expression of cGAS determines tumor immunogenicity and makes tumors hot. These findings are relevant for genotoxic and immune therapies for cancer.